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1.
Neuron ; 109(7): 1188-1201.e7, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33577748

RESUMO

Proprioception is essential for behavior and provides a sense of our body movements in physical space. Proprioceptor organs are thought to be only in the periphery. Whether the central nervous system can intrinsically sense its own movement remains unclear. Here we identify a segmental organ of proprioception in the adult zebrafish spinal cord, which is embedded by intraspinal mechanosensory neurons expressing Piezo2 channels. These cells are late-born, inhibitory, commissural neurons with unique molecular and physiological profiles reflecting a dual sensory and motor function. The central proprioceptive organ locally detects lateral body movements during locomotion and provides direct inhibitory feedback onto rhythm-generating interneurons responsible for the central motor program. This dynamically aligns central pattern generation with movement outcome for efficient locomotion. Our results demonstrate that a central proprioceptive organ monitors self-movement using hybrid neurons that merge sensory and motor entities into a unified network.


Assuntos
Retroalimentação Sensorial/fisiologia , Movimento/fisiologia , Propriocepção/fisiologia , Peixe-Zebra/fisiologia , Animais , Geradores de Padrão Central/fisiologia , Feminino , Interneurônios/fisiologia , Canais Iônicos/fisiologia , Locomoção/fisiologia , Masculino , Mecanotransdução Celular , Neurônios Motores/fisiologia , Rede Nervosa/citologia , Rede Nervosa/fisiologia , RNA/genética , Células Receptoras Sensoriais/fisiologia , Medula Espinal/diagnóstico por imagem , Medula Espinal/fisiologia , Tomografia Computadorizada por Raios X , Proteínas de Peixe-Zebra/fisiologia
2.
Nucleic Acids Res ; 49(4): 2027-2043, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33476374

RESUMO

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.


Assuntos
Processamento Alternativo , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Retina/embriologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Apoptose , Sistemas CRISPR-Cas , Sobrevivência Celular , Dano ao DNA , Reparo do DNA , Éxons , Técnicas de Inativação de Genes , Pontos de Checagem da Fase M do Ciclo Celular , Células-Tronco Neurais/citologia , Neurônios Retinianos/citologia , Neurônios Retinianos/metabolismo , Fuso Acromático/ultraestrutura , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
3.
Dev Biol ; 469: 54-67, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971120

RESUMO

Tie1 is a receptor tyrosine kinase expressed in endothelial cells, where it modulates Angiopoietin/Tie2 signaling. Previous studies have shown that mouse Tie1 mutants exhibit severe cardiovascular defects; however, much remains to be learned about the role of Tie1, especially during cardiac development. To further understand Tie1 function, we generated a zebrafish tie1 mutant line. Homozygous mutant embryos display reduced endothelial and endocardial cell numbers and reduced heart size. Live imaging and ultrastructural analyses at embryonic stages revealed increased cardiac jelly thickness as well as cardiomyocyte defects, including a loss of sarcomere organization and altered cell shape. Transcriptomic profiling of embryonic hearts uncovered the downregulation of tll1, which encodes a Tolloid-like protease, in tie1-/- compared with wild-type siblings. Using mRNA injections into one-cell stage embryos, we found that tll1 overexpression could partially rescue the tie1 mutant cardiac phenotypes including the endocardial and myocardial cell numbers as well as the cardiac jelly thickness. Altogether, our results indicate the importance of a Tie1-Tolloid-like 1 axis in paracrine signaling during cardiac development.


Assuntos
Coração/embriologia , Metaloproteases Semelhantes a Toloide/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Células Endoteliais/citologia , Endotélio Vascular/citologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Cardiopatias Congênitas/genética , Morfogênese , Mutação , Miócitos Cardíacos/citologia , Receptor de TIE-1/genética , Receptor de TIE-1/fisiologia , Metaloproteases Semelhantes a Toloide/genética , Transcriptoma , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
4.
Nat Commun ; 11(1): 5319, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-33087700

RESUMO

Arterial networks enlarge in response to increase in tissue metabolism to facilitate flow and nutrient delivery. Typically, the transition of a growing artery with a small diameter into a large caliber artery with a sizeable diameter occurs upon the blood flow driven change in number and shape of endothelial cells lining the arterial lumen. Here, using zebrafish embryos and endothelial cell models, we describe an alternative, flow independent model, involving enlargement of arterial endothelial cells, which results in the formation of large diameter arteries. Endothelial enlargement requires the GEF1 domain of the guanine nucleotide exchange factor Trio and activation of Rho-GTPases Rac1 and RhoG in the cell periphery, inducing F-actin cytoskeleton remodeling, myosin based tension at junction regions and focal adhesions. Activation of Trio in developing arteries in vivo involves precise titration of the Vegf signaling strength in the arterial wall, which is controlled by the soluble Vegf receptor Flt1.


Assuntos
Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Remodelação Vascular/fisiologia , Animais , Animais Geneticamente Modificados , Tamanho Celular , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Cardiovasculares , Fator de Crescimento Placentário/genética , Fator de Crescimento Placentário/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Remodelação Vascular/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/fisiologia
5.
Gene ; 751: 144761, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32407768

RESUMO

The ubiquitin specific peptidase (USP) family is involved in many life processes, of which antiviral is also an important basic function. One of the more important ways is to activate interferon. In this study, we reported the antiviral function of the ubiquitin specific peptidase 5(USP5) gene in zebrafish. Evolutionary and comparative protein sequence analysis of the USP5 was performed. The localization of USP5 in FHM cells cytoplasm was determined. Overexpression of USP5 significantly evoked higher expression of mRNA that encode IFNφ1 and ISGs, the promoteractivities of IFNφ1 and IFNstimulated response element (ISRE) were augmented likewise. USP5 was also able to enhance the expression of RIG-I and activate higher levels of IFNφ1 stimulated by Poly (I: C). Viral infection and interference tests demonstrated that USP5 inhibited the replication of SVCV in vitro. In summary, this study reveals that USP5 is able to activate higher levels of interferon by increasing RIG-I protein levels, and thus implement antivirus functions.


Assuntos
Proteases Específicas de Ubiquitina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Interferons/biossíntese , Interferons/genética , Rhabdoviridae/fisiologia , Alinhamento de Sequência , Proteases Específicas de Ubiquitina/genética , Replicação Viral , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Peixe-Zebra/virologia , Proteínas de Peixe-Zebra/genética
6.
Gene ; 744: 144632, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32240777

RESUMO

IGF2BPs, a subclass of RNA-binding proteins, regulate cellular differentiation, proliferation and migration during multiple organs development, but their functions in liver development still remain unclear. Here, in this study, whole-mount in situ hybridization showed that igf2bp1 was constantly and stably expressed at early stages of embryo development in zebrafish. Both the morpholino-induced knockdown and CRISPR/Cas9-mediated knockout of igf2bp1 led to a reduced-size liver phenotype. Further analysis revealed that igf2bp1 is required for hepatic outgrowth, but not for hepatoblast specification and budding. Deficiency of igf2bp1 resulted in reduced cell proliferation, but had no effect on apoptosis. Therefore, we concluded that igf2bp1 is a critical factor to regulate hepatic outgrowth via cell proliferation during early liver development in zebrafish.


Assuntos
Fígado/embriologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Proliferação de Células , Hepatócitos/citologia , Fígado/anatomia & histologia , Fígado/metabolismo , Morfolinos , Tamanho do Órgão , Fenótipo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
J Neurosci ; 40(15): 3063-3074, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32139583

RESUMO

The cerebellum influences motor control through Purkinje target neurons, which transmit cerebellar output. Such output is required, for instance, for larval zebrafish to learn conditioned fictive swimming. The output cells, called eurydendroid neurons (ENs) in teleost fish, are inhibited by Purkinje cells and excited by parallel fibers. Here, we investigated the electrophysiological properties of glutamatergic ENs labeled by the transcription factor olig2. Action potential firing and synaptic responses were recorded in current clamp and voltage clamp from olig2+ neurons in immobilized larval zebrafish (before sexual differentiation) and were correlated with motor behavior by simultaneous recording of fictive swimming. In the absence of swimming, olig2+ ENs had basal firing rates near 8 spikes/s, and EPSCs and IPSCs were evident. Comparing Purkinje firing rates and eurydendroid IPSC rates indicated that 1-3 Purkinje cells converge onto each EN. Optogenetically suppressing Purkinje simple spikes, while preserving complex spikes, suggested that eurydendroid IPSC size depended on presynaptic spike duration rather than amplitude. During swimming, EPSC and IPSC rates increased. Total excitatory and inhibitory currents during sensory-evoked swimming were both more than double those during spontaneous swimming. During both spontaneous and sensory-evoked swimming, the total inhibitory current was more than threefold larger than the excitatory current. Firing rates of ENs nevertheless increased, suggesting that the relative timing of IPSCs and EPSCs may permit excitation to drive additional eurydendroid spikes. The data indicate that olig2+ cells are ENs whose activity is modulated with locomotion, suiting them to participate in sensorimotor integration associated with cerebellum-dependent learning.SIGNIFICANCE STATEMENT The cerebellum contributes to movements through signals generated by cerebellar output neurons, called eurydendroid neurons (ENs) in fish (cerebellar nuclei in mammals). ENs receive sensory and motor signals from excitatory parallel fibers and inhibitory Purkinje cells. Here, we report electrophysiological recordings from ENs of larval zebrafish that directly illustrate how synaptic inhibition and excitation are integrated by cerebellar output neurons in association with motor behavior. The results demonstrate that inhibitory and excitatory drive both increase during fictive swimming, but inhibition greatly exceeds excitation. Firing rates nevertheless increase, providing evidence that synaptic integration promotes cerebellar output during locomotion. The data offer a basis for comparing aspects of cerebellar coding that are conserved and that diverge across vertebrates.


Assuntos
Cerebelo/fisiologia , Neurônios/fisiologia , Fator de Transcrição 2 de Oligodendrócitos/fisiologia , Natação/fisiologia , Sinapses/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Potenciais de Ação/fisiologia , Animais , Animais Geneticamente Modificados , Fenômenos Eletrofisiológicos/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Larva , Optogenética , Técnicas de Patch-Clamp , Células de Purkinje/fisiologia
8.
Development ; 147(6)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179574

RESUMO

Precise temporal coordination of signaling processes is pivotal for cellular differentiation during embryonic development. A vast number of secreted molecules are produced and released by cells and tissues, and travel in the extracellular space. Whether they induce a signaling pathway and instruct cell fate, however, depends on a complex network of regulatory mechanisms, which are often not well understood. The conserved bilateral left-right asymmetrically formed habenulae of the zebrafish are an excellent model for investigating how signaling control facilitates the generation of defined neuronal populations. Wnt signaling is required for habenular neuron type specification, asymmetry and axonal connectivity. The temporal regulation of this pathway and the players involved have, however, have remained unclear. We find that tightly regulated temporal restriction of Wnt signaling activity in habenular precursor cells is crucial for the diversity and asymmetry of habenular neuron populations. We suggest a feedback mechanism whereby the tumor suppressor Wnt inhibitory factor Wif1 controls the Wnt dynamics in the environment of habenular precursor cells. This mechanism might be common to other cell types, including tumor cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Padronização Corporal/genética , Habenula/embriologia , Neurogênese/genética , Neurônios/fisiologia , Proteínas Repressoras/fisiologia , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Encéfalo/embriologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Dominância Cerebral/genética , Embrião não Mamífero , Habenula/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Development ; 147(4)2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32001440

RESUMO

Sex determination and differentiation are complex processes controlled by many different factors; however, the relationships among these factors are poorly understood. Zebrafish gonadal differentiation exhibits high plasticity involving multiple factors and pathways, which provides an excellent model for investigating the interactions between them. Ovarian aromatase (cyp19a1a) and dmrt1 are key factors in directing vertebrate ovary and testis differentiation, respectively. Knockout of zebrafish cyp19a1a leads to all-male offspring, whereas the loss of dmrt1 results in a female-biased sex ratio. In the present study, we established dmrt1-/- ;cyp19a1a-/- double mutant zebrafish and discovered that the introduction of the dmrt1 mutation into the cyp19a1a mutant could rescue the all-male phenotype of the latter. Interestingly, despite the lack of aromatase/estrogens, the follicles in the ovary of the rescued cyp19a1a mutant could develop normally up to the previtellogenic stage. Further evidence suggested the ovarian aromatase directed ovarian differentiation by suppressing dmrt1 expression via nuclear estrogen receptors (nERs). Our results provide solid evidence for an interaction between cyp19a1a and dmrt1 in zebrafish gonadal differentiation, and for the dispensability of estrogens in controlling early folliculogenesis.


Assuntos
Aromatase/genética , Aromatase/fisiologia , Folículo Ovariano/embriologia , Testículo/embriologia , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Alelos , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Estrogênios/fisiologia , Feminino , Técnicas de Inativação de Genes , Genótipo , Heterozigoto , Masculino , Mutação , Fenótipo , Receptores Estrogênicos/fisiologia , Processos de Determinação Sexual , Diferenciação Sexual , Peixe-Zebra
10.
Development ; 147(4)2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31988185

RESUMO

Organogenesis requires precise interactions between a developing tissue and its environment. In vertebrates, the developing eye is surrounded by a complex extracellular matrix as well as multiple mesenchymal cell populations. Disruptions to either the matrix or periocular mesenchyme can cause defects in early eye development, yet in many cases the underlying mechanism is unknown. Here, using multidimensional imaging and computational analyses in zebrafish, we establish that cell movements in the developing optic cup require neural crest. Ultrastructural analysis reveals that basement membrane formation around the developing eye is also dependent on neural crest, but only specifically around the retinal pigment epithelium. Neural crest cells produce the extracellular matrix protein nidogen: impairing nidogen function disrupts eye development, and, strikingly, expression of nidogen in the absence of neural crest partially restores optic cup morphogenesis. These results demonstrate that eye formation is regulated in part by extrinsic control of extracellular matrix assembly.This article has an associated 'The people behind the papers' interview.


Assuntos
Membrana Basal/embriologia , Olho/embriologia , Crista Neural/embriologia , Alelos , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/fisiologia , Movimento Celular , Eletroforese Capilar , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Fatores de Transcrição Forkhead/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Mesoderma/embriologia , Microscopia Eletrônica de Transmissão , Morfogênese , Mutação , Crista Neural/citologia , Organogênese , Retina/embriologia , Epitélio Pigmentado da Retina/embriologia , Transdução de Sinais , Fator de Transcrição AP-2/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologia
11.
Development ; 147(1)2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31852685

RESUMO

In order to efficiently derive hematopoietic stem cells (HSCs) from pluripotent precursors, it is crucial to understand how mesodermal cells acquire hematopoietic and endothelial identities: two divergent, but closely related, cell fates. Although Npas4 has been recently identified as a conserved master regulator of hemato-vascular development, the molecular mechanisms underlying cell fate divergence between hematopoietic and vascular endothelial cells are still unclear. Here, we show in zebrafish that mesodermal cell differentiation into hematopoietic and vascular endothelial cells is regulated by Junctional adhesion molecule 3b (Jam3b) via two independent signaling pathways. Mutation of jam3b led to a reduction in npas4l expression in the posterior lateral plate mesoderm and defects in both hematopoietic and vascular development. Mechanistically, we show that Jam3b promotes endothelial specification by regulating npas4l expression through repression of the Rap1a-Erk signaling cascade. Jam3b subsequently promotes hematopoietic development, including HSCs, by regulating lrrc15 expression in endothelial precursors through the activation of an integrin-dependent signaling cascade. Our data provide insight into the divergent mechanisms for instructing hematopoietic or vascular fates from mesodermal cells.


Assuntos
Sistema Cardiovascular/embriologia , Hematopoese , Receptores de Superfície Celular/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sistema Cardiovascular/citologia , Células Endoteliais/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Sistema de Sinalização das MAP Quinases , Mesoderma/embriologia , Receptores de Superfície Celular/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Dev Biol ; 458(1): 98-105, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31682806

RESUMO

Attempts to constitutively knockout HTT in rodents resulted in embryonic lethality, curtailing efforts to study HTT function later in development. Here we show that HTT is dispensable for early zebrafish development, contrasting published zebrafish morpholino experiment results. Homozygous HTT knockouts were embryonically viable and appeared developmentally normal through juvenile stages. Comparison of adult fish revealed significant reduction in body size and fitness in knockouts compared to hemizygotes and wildtype fish, indicating an important role for wildtype HTT in postnatal development. Our zebrafish model provides an opportunity to understand the function of wildtype HTT later in development.


Assuntos
Modelos Animais , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Tamanho Corporal , Sistemas CRISPR-Cas , Sequência Conservada , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Edição de Genes , Técnicas de Inativação de Genes , Estudos de Associação Genética , Aptidão Genética , Humanos , Proteína Huntingtina/química , Morfolinos/farmacologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurulação/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
13.
J Neurosci ; 40(1): 143-158, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31685652

RESUMO

Down syndrome cell adhesion molecules (dscam and dscaml1) are essential regulators of neural circuit assembly, but their roles in vertebrate neural circuit function are still mostly unexplored. We investigated the functional consequences of dscaml1 deficiency in the larval zebrafish (sexually undifferentiated) oculomotor system, where behavior, circuit function, and neuronal activity can be precisely quantified. Genetic perturbation of dscaml1 resulted in deficits in retinal patterning and light adaptation, consistent with its known roles in mammals. Oculomotor analyses revealed specific deficits related to the dscaml1 mutation, including severe fatigue during gaze stabilization, reduced saccade amplitude and velocity in the light, greater disconjugacy, and impaired fixation. Two-photon calcium imaging of abducens neurons in control and dscaml1 mutant animals confirmed deficits in saccade-command signals (indicative of an impairment in the saccadic premotor pathway), whereas abducens activation by the pretectum-vestibular pathway was not affected. Together, we show that loss of dscaml1 resulted in impairments in specific oculomotor circuits, providing a new animal model to investigate the development of oculomotor premotor pathways and their associated human ocular disorders.SIGNIFICANCE STATEMENT Dscaml1 is a neural developmental gene with unknown behavioral significance. Using the zebrafish model, this study shows that dscaml1 mutants have a host of oculomotor (eye movement) deficits. Notably, the oculomotor phenotypes in dscaml1 mutants are reminiscent of human ocular motor apraxia, a neurodevelopmental disorder characterized by reduced saccade amplitude and gaze stabilization deficits. Population-level recording of neuronal activity further revealed potential subcircuit-specific requirements for dscaml1 during oculomotor behavior. These findings underscore the importance of dscaml1 in the development of visuomotor function and characterize a new model to investigate potential circuit deficits underlying human oculomotor disorders.


Assuntos
Movimentos Oculares/fisiologia , Adaptação Ocular/genética , Adaptação Ocular/fisiologia , Células Amácrinas/fisiologia , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , Moléculas de Adesão Celular/fisiologia , Movimentos Oculares/genética , Fixação Ocular/genética , Fixação Ocular/fisiologia , Larva , Locomoção , Fadiga Muscular , Mutação , Músculos Oculomotores/crescimento & desenvolvimento , Músculos Oculomotores/fisiopatologia , Retina/crescimento & desenvolvimento , Retina/ultraestrutura , Movimentos Sacádicos/genética , Movimentos Sacádicos/fisiologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/fisiologia
14.
Development ; 146(24)2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31784460

RESUMO

Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the RNA polymerase-associated factor 1 complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; alyron mutant embryos carrying a null mutation in paf1 were analyzed in detail. In the absence of zygotic paf1 function, definitive premigratory NC progenitors arise but fail to maintain expression of the sox10 specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of positive transcription elongation factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.


Assuntos
Linhagem da Célula/genética , Células-Tronco Multipotentes/fisiologia , Crista Neural/citologia , Células-Tronco Neurais/fisiologia , Proteínas Nucleares/fisiologia , Fator B de Elongação Transcricional Positiva/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Diferenciação Celular/genética , Quinase 9 Dependente de Ciclina/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Multipotentes/citologia , Complexos Multiproteicos/genética , Complexos Multiproteicos/fisiologia , Crista Neural/fisiologia , Células-Tronco Neurais/citologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fator B de Elongação Transcricional Positiva/antagonistas & inibidores , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
15.
Development ; 146(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31666235

RESUMO

Connexin 39.4 (Cx39.4) and connexin 41.8 (Cx41.8), two gap-junction proteins expressed in both melanophores and xanthophores, are crucial for the intercellular communication among pigment cells that is necessary for generating the stripe pigment pattern of zebrafish. We have previously characterized the gap-junction properties of Cx39.4 and Cx41.8, but how these proteins contribute to stripe formation remains unclear; this is because distinct types of connexins potentially form heteromeric gap junctions, which precludes accurate elucidation of individual connexin functions in vivo Here, by arranging Cx39.4 and Cx41.8 expression in pigment cells, we have identified the simplest gap-junction network required for stripe generation: Cx39.4 expression in melanophores is required but expression in xanthophores is not necessary for stripe patterning, whereas Cx41.8 expression in xanthophores is sufficient for the patterning, and Cx41.8 expression in melanophores might stabilize the stripes. Moreover, patch-clamp recordings revealed that Cx39.4 gap junctions exhibit spermidine-dependent rectification property. Our results suggest that Cx39.4 facilitates the crucial cell-cell interactions between melanophores and xanthophores that mediate a unidirectional activation-signal transfer from xanthophores to melanophores, which is essential for melanophore survival.


Assuntos
Padronização Corporal , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Melanóforos/fisiologia , Pigmentação , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Comunicação Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Eletrofisiologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Mutação , Fenótipo , Plasmídeos , Transdução de Sinais , Espermidina/química , Transgenes , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/metabolismo
16.
Dev Dyn ; 248(12): 1243-1256, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31566834

RESUMO

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a rare congenital disorder characterized by a lack of blood vessel growth to the periphery of the retina with secondary fibrovascular proliferation at the vascular-avascular junction. These structurally abnormal vessels cause leakage and hemorrhage, while the fibroproliferative scarring results in retinal dragging, detachment and blindness. Mutations in the FZD4 gene represent one of the most common causes of FEVR. METHODS: A loss of function mutation resulting from a 10-nucleotide insertion into exon 1 of the zebrafish fzd4 gene was generated using transcription activator-like effector nucleases (TALENs). Structural and functional integrity of the retinal vasculature was examined by fluorescent microscopy and optokinetic responses. RESULTS: Zebrafish retinal vasculature is asymmetrically distributed along the dorsoventral axis, with active vascular remodeling on the ventral surface of the retina throughout development. fzd4 mutants exhibit disorganized ventral retinal vasculature with discernable tubular fusion by week 8 of development. Furthermore, fzd4 mutants have impaired optokinetic responses requiring increased illumination. CONCLUSION: We have generated a visually impaired zebrafish FEVR model exhibiting abnormal retinal vasculature. These fish provide a tractable system for studying vascular biology in retinovascular disorders, and demonstrate the feasibility of using zebrafish for evaluating future FEVR genes identified in humans.


Assuntos
Receptores Frizzled/fisiologia , Retina/patologia , Vasos Retinianos/patologia , Remodelação Vascular/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Modelos Animais de Doenças , Embrião não Mamífero , Vitreorretinopatias Exsudativas Familiares/diagnóstico , Vitreorretinopatias Exsudativas Familiares/genética , Vitreorretinopatias Exsudativas Familiares/patologia , Estudos de Viabilidade , Receptores Frizzled/genética , Humanos , Neovascularização Patológica/embriologia , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Retina/diagnóstico por imagem , Retina/embriologia , Retina/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/patologia , Vasos Retinianos/embriologia , Vasos Retinianos/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
17.
Cells ; 8(9)2019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480347

RESUMO

There are 19 Wnt genes in mammals that belong to 12 subfamilies. Wnt signaling pathways participate in regulating numerous homeostatic and developmental processes in animals. However, the function of Wnt10b in fatty acid synthesis remains unclear in fish species. In the present study, we uncovered the role of the Wnt10b signaling pathway in the regulation of fatty acid synthesis in the muscle of zebrafish. The gene of Wnt10b was overexpressed in the muscle of zebrafish using pEGFP-N1-Wnt10b vector injection, which significantly decreased the expression of glycogen synthase kinase 3ß (GSK-3ß), but increased the expression of ß-catenin, peroxisome proliferators-activated receptor γ (PPARγ), and CCAAT/enhancer binding protein α (C/EBPα). Moreover, the activity and mRNA expression of key lipogenic enzymes ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS), and the content of non-esterified fatty acids (NEFA), total cholesterol (TC), and triglyceride (TG) were also significantly decreased. Furthermore, interference of the Wnt10b gene significantly inhibited the expression of ß-catenin, PPARγ, and C/EBPα, but significantly induced the expression of GSK-3ß, FAS, ACC, and ACL. The content of NEFA, TC, and TG as well as the activity of FAS, ACC, and ACL significantly increased. Thus, our results showed that Wnt10b participates in regulating fatty acid synthesis via ß-catenin, C/EBPα and PPARγ in the muscle of zebrafish.


Assuntos
Ácidos Graxos/metabolismo , Músculos/metabolismo , Proteínas Wnt/fisiologia , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , PPAR gama/metabolismo , Proteínas Wnt/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , beta Catenina/metabolismo
18.
Neuron ; 104(2): 271-289.e13, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31515109

RESUMO

Mutations in one SETD5 allele are genetic causes of intellectual disability and autistic spectrum disorders. However, the mechanisms by which SETD5 regulates brain development and function remain largely elusive. Herein, we found that Setd5 haploinsufficiency impairs the proliferative dynamics of neural progenitors and synaptic wiring of neurons, ultimately resulting in behavioral deficits in mice. Mechanistically, Setd5 inactivation in neural stem cells, zebrafish, and mice equally affects genome-wide levels of H3K36me3 on active gene bodies. Notably, we demonstrated that SETD5 directly deposits H3K36me3, which is essential to allow on-time RNA elongation dynamics. Hence, Setd5 gene loss leads to abnormal transcription, with impaired RNA maturation causing detrimental effects on gene integrity and splicing. These findings identify SETD5 as a fundamental epigenetic enzyme controlling the transcriptional landscape in neural progenitors and their derivatives and illuminate the molecular events that connect epigenetic defects with neuronal dysfunctions at the basis of related human diseases.


Assuntos
Encéfalo/embriologia , Cromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Metiltransferases/genética , Proteínas de Peixe-Zebra/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Cognição , Epigênese Genética , Histona Metiltransferases/genética , Metiltransferases/fisiologia , Camundongos , Mutação , Células-Tronco Neurais/metabolismo , Processamento de RNA/genética , RNA-Seq , Comportamento Social , Elongação da Transcrição Genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
19.
Dev Biol ; 455(2): 473-484, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31394080

RESUMO

Intestinal tract development is a coordinated process involving signaling among the progenitors and developing cells from all three germ layers. Development of endoderm-derived intestinal epithelium has been shown to depend on epigenetic modifications, but whether that is also the case for intestinal tract cell types from other germ layers remains unclear. We found that functional loss of a DNA methylation machinery component, ubiquitin-like protein containing PHD and RING finger domains 1 (uhrf1), leads to reduced numbers of ectoderm-derived enteric neurons and severe disruption of mesoderm-derived intestinal smooth muscle. Genetic chimeras revealed that Uhrf1 functions both cell-autonomously in enteric neuron precursors and cell-non-autonomously in surrounding intestinal cells, consistent with what is known about signaling interactions between these cell types that promote one another's development. Uhrf1 recruits the DNA methyltransferase Dnmt1 to unmethylated DNA during replication. Dnmt1 is also expressed in enteric neurons and smooth muscle progenitors. dnmt1 mutants have fewer enteric neurons and disrupted intestinal smooth muscle compared to wildtypes. Because dnmt1;uhrf1 double mutants have a similar phenotype to dnmt1 and uhrf1 single mutants, Dnmt1 and Uhrf1 must function together during enteric neuron and intestinal muscle development. This work shows that genes controlling epigenetic modifications are important to coordinate intestinal tract development, provides the first demonstration that these genes influence development of the ENS, and advances uhrf1 and dnmt1 as potential new Hirschsprung disease candidates.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/fisiologia , Sistema Nervoso Entérico/embriologia , Epigênese Genética , Intestinos/embriologia , Transativadores/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Quimera , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Intestinos/citologia , Intestinos/inervação , Masculino , Músculo Liso/embriologia , Mutação , Neurônios , Transativadores/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
20.
Development ; 146(16)2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31405994

RESUMO

Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30 days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.


Assuntos
Fatores de Transcrição Kruppel-Like/fisiologia , Neurogênese , Células Ganglionares da Retina/fisiologia , Animais , Ciclo Celular , Feminino , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração Nervosa , Células-Tronco Neurais/fisiologia , Ratos , Fator de Transcrição Brn-3A/metabolismo , Fator de Transcrição Brn-3B/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologia
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